Farnesyl pyrophosphate synthase protein, nucleic acid and promoter region thereof

ABSTRACT

The promoter region of the farnesyl pyrophosphate synthase gene that was expressed in the hop luplin gland in a specific manner was elucidated based on the genomic DNA of the hop farnesyl pyrophosphate synthase gene having the nucleotide sequence set forth in SEQ ID NO:2, the cDNA of the hop farnesyl pyrophosphate synthase gene having the nucleotide sequence set forth in SEQ ID NO:3, and the nucleotide sequence information on the genomic DNA and the cDNA. It will reveal the gene involved in the biosynthesis of secondary metabolites in a hop as well as the nucleotide sequence of the promoter gene that functions in the hop luplin gland in a tissue-specific manner. This will allow for the transformation of the hop by gene manipulations and the in vitro synthesis of the hop secondary metabolites.

TECHNICAL FIELD

This invention relates to farnesyl pyrophosphate (FPP) synthase genes ofhop and their promoter regions.

BACKGROUND ART

Plants produce and accumulate within their bodies, numerous kinds of lowmolecular weight organic compounds such as terpenoids, alkaloids,phenolics, and saponins. It was initially thought that these compoundswere not directly responsible for the life maintenance of organisms andhave only auguxilliary functions; therefore, they were referred to as“secondary metabolites.”

However, in recent years it has been beginning to be understood thatthese secondary metabolites function as substances responsible for thecell differentiation or the defense against exogenous factors.Concurrently, the methods of utilization are being found in the broadfields of taste products, drugs and pigments. Not to mention theagricultural field, their utility is catching attention in broad fields.

Because such secondary metabolites are valuable in being industriallyutilized, the elucidation of their formation process in plant cells hasprogressed and presently it has been shown that the substances aresynthesized through a complex cascade involving a large number ofenzymes. Direct Extraction from plants is, however, needed to obtainsuch substances. In those cases the quantities that can be isolated atone time are very small, resulting in high cost. Therefore, it has beendesired that in vitro synthetic methods using gene manipulations orcultured cells be developed.

Farnesyl pyrophosphate synthase is known as an enzyme that is involvedin the synthetic cascade of the secondary metabolites in plants.Farnesyl pyrophosphate synthase is an enzyme that is involved in themetabolism of isoprenoids which forms the basis for a variety ofsubstances in plants such as pigments, odorants, phytohormones,phytoalexins, and defense substances against pests (Plant Biochemistry &Molecular Biology, Hans-Walter Heldt, Oxford University Press, pp.360-376, 1997). It has been shown that farnesyl pyrophosphate synthasecatalyzes the reaction converting isopentenyl pyrophosphate into geranylpyrophosphate by adding dimethylallyl pyrophosphate thereto as well ascatalyzes the reaction converting said geranyl pyrophosphate intofarnesyl pyrophosphate by adding isopentenyl pyrophosphate thereto.

Hop is a principal material to give beer refreshing bitter taste andflavor. It is beginning to be understood that the secondary metabolitesare secreted in large quantities in luplin gland contained in the coneof the hop and these secondary metabolites greatly contribute to thebitter taste and flavor of beer. Further, in recent years it has beenshown that the secondary metabolites of the hop possess pharmacologicalactions (for example, Biosci. Biotech. Biochem., 61 (1), 158, 1997).Under such circumstances, a variety of breeding improvements are beingmade to the hop with an emphasis on the secondary metabolitesaccumulated in the luplin gland such as bitter substances and essentialoil constituents.

However, the hop is a dioecious plant. Especially, the male plant doesnot beer cones that serves as the beer material and thus is not regardedimportant commercially. Very little study, therefore, has been carriedout and the genetic traits that will be useful for fermentation havebeen hardly elucidated. For these reasons, conventional breeding methodsthrough crossing largely rely on experience and intuition. The presentsituation is that the brewing qualities are totally unpredictable untilthe cones have grown. Accordingly, it is strongly desired that thefarnesyl pyrophosphate synthase gene be isolated from a hop and that thecontrol of the secondary metabolites in the hop and in vitro syntheticmethods be established according to an approach using genemanipulations.

The breeding methods utilizing genetic engineering such astransformation techniques and molecular selection techniques arebecoming possibilities in various plants. In these methods, moreobjective and efficient breeding is possible as compared to theconventional breeding methods which largely rely on experience andintuition. More specifically, the transformation technique is one inwhich an exogenous gene is introduced into a plant cell to have itexpressed and the trait to be desirably incorporated is directlyintroduced into the cell. In order to have the exogenous gene expressed,the following method may be employed: an objective structural gene and aterminator operable in a plant cell are linked to an operable promotercapable of regulating the expression of the gene in the plant cell andthe linkage is introduced into the plant cell. For a promoter that isfrequently used at the experimental level, there are mentioned, amongothers, a CaMV 35S promoter, a nopaline synthase gene promoter both ofwhich can express transgenes in relatively large kinds of plantsregardless of their tissues (Sanders P. R. et al., Nucleic Acid Res, 15(1987) 1543-1558). However, when the aforementioned promoters are usedto express the transgenes in all the tissues, some transgenes may doharm to the growth of the plants. It is, therefore, strongly desiredthat tissue-specific promoters capable of expressing exogenous genes inthe objective tissue be isolated.

DISCLOSURE OF THE INVENTION

This invention has been made in view of the problems that are inherentin the aforementioned prior art; it aims at elucidating the genesinvolved in the biosynthesis of secondary metabolites in a hop and thenucleotide sequences (base sequences) of the promoters operable in atissue-specific manner in the luplin gland of the hop as well as aims atallowing for the transformation of the hop by gene manipulations and thein vitro synthesis of the secondary metabolite of the hop.

As a result of having pursued diligent investigations to accomplish theabove-stated objects, the present inventors found farnesyl pyrophosphatesynthase genes and their promoter genes, which led to the completion ofthis invention: the farnesyl pyrophosphate synthase genes were stronglyexpressed in the luplin gland of the hop and were involved in thebiosynthesis of secondary metabolites.

Specifically, according to this invention there are provided theproteins described in 1-2 below:

1. A protein having the amino acid sequence set forth in SEQ ID NO:1 inthe Sequence Listing.

2. A protein having an amino acid sequence derivable from the deletionor the substitution of one or more amino acids in the amino acidsequence set forth in SEQ ID NO:1 in the Sequence Listing, or from theaddition of one or more amino acids to the amino acid sequence set forthin SEQ ID NO:1 in the Sequence Listing, said protein possessing thefarnesyl pyrophosphate synthase activity.

Also, according to this invention there are provided the nucleic acidsdescribed in 3-10 below:

3. A nucleic acid encoding a protein having the amino acid sequence setforth in SEQ ID NO:1 in the Sequence Listing.

4. A nucleic acid having the nucleotide sequence set forth in SEQ IDNO:3 in the Sequence Listing.

5. A nucleic acid comprising a part of the nucleotide sequence set forthin SEQ ID NO:3 in the Sequence Listing.

6. A nucleic acid that hybridizes to a nucleic acid having thenucleotide sequence set forth in SEQ ID NO:3 in the Sequence Listing orto a complementary nucleic acid thereof under stringent conditions, saidnucleic acid encoding a protein possessing the farnesyl pyrophosphatesynthase activity.

7. A nucleic acid having the nucleotide sequence set forth in SEQ IDNO:2 in the Sequence Listing.

8. A nucleic acid comprising a part of the nucleotide sequence set forthin SEQ ID NO:2 in the Sequence Listing.

9. A nucleic acid having a nucleotide sequence of from base No. 1 tobase No. 1886 in the nucleotide sequence set forth in SEQ ID NO:2 in theSequence Listing.

10. A nucleic acid that hybridizes to a nucleic acid having thenucleotide sequence described in 7 or 8 above or to a complementarynucleic acid thereof under stringent conditions, said nucleic acidpossessing promoter activity.

The use of these proteins or nucleic acids will then enable thetransformation of a hop by gene manipulations as well as enable the invitro synthesis of the secondary metabolites of a hop.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing nucleic acid fragments obtained from theisolation process of a farnesyl pyrophosphate synthase gene of thisinvention.

FIG. 2 is a schematic diagram showing the principle of Inverse PCR usedin the invention.

FIG. 3 is a schematic diagram showing the principle of Casette-ligationmediate PCR used in the invention.

FIG. 4 is a representation showing the developed image of thin layerchromatography used when the activity of the farnesyl pyrophosphatesynthase of the invention was determined.

FIG. 5 is a photograph of Northern analysis that confirmed theexpression of the farnesyl pyrophosphate synthase gene of the invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The preferred embodiments of this invention will be described in detailhereafter by referring to the drawings when necessary.

As used in the invention, “nucleic acid” refers to DNA, RNA, or apolynucleotide that may be a derivatized active DNA or RNA among others.Preferred are DNA and/or RNA. In this case, the forms of the nucleicacid include genomic DNA, cDNA and mRNA, for example.

As used in the invention, “hybridize under stringent conditions” meansthat two nucleic acid fragments hybridize to each other under thehybridization conditions as described in Sambrook, J. et al.,“Expression of Cloned Genes in E. coli,” Molecular Cloning: A LaboratoryManual (1989) Cold Spring Harbor Laboratory Press, New York, USA,9.47-9.62 and 11.45-11.61.

More specifically, “stringent conditions” means that after hybridizationin 6.0×SSC at about 45° C., washing is to be done in 2.0×SSC at 50° C.,for example. For the purpose of selection of stringency, the saltconcentration in the washing step can be selected to be from about2.0×SSC at 50° C. under low stringency to about 0.1×SSC at 50° C. underhigh stringency. Furthermore, the temperature of washing step can beincreased from room temperature under low stringency conditions (about22° C.) to about 65° C. under high stringency conditions.

As used in the invention, “promoter” refers to a nucleotide sequencepresent in DNA that is a signaling sequence functioning to governdirecting the initiation and termination of RNA synthesis(transcription) or directing the regulation of its frequency.

As used in the invention, “promoter activity” refers to the function ofinitiating, terminating, and regulating transcription by the promoter asdescribed above.

The hop farnesyl pyrophosphate synthase of this invention will be firstdescribed.

The protein of this invention is a hop farnesyl pyrophosphate synthaseprotein having the amino acid sequence with 342 amino acid residues, asset forth in SEQ ID NO:1 in the Sequence Listing.

Also, the protein of the invention may be a protein having an amino acidsequence derivable from the deletion or the substitution of one or moreamino acids in the amino acid sequence set forth in SEQ ID NO:1 in theSequence Listing, or from the addition of one or more amino acids to theamino acid sequence set forth in SEQ ID NO:1 in the Sequence Listing,insofar as it possesses the farnesyl pyrophosphate synthase activity.

Further, since sugar chains may be appended to a large number ofproteins, the addition of sugar chains can be adjusted by subjecting oneor more amino acids to conversion. Therefore, proteins for which theaddition of sugar chains within the amino acid sequence set forth in SEQID NO:1 in the Sequence Listing has been adjusted are also embraced bythe proteins of this invention insofar as they possess the farnesylpyrophosphate synthase activity as described above.

Further, nucleic acids having nucleotide sequences encoding theaforementioned farnesyl pyrophosphate synthase proteins are alsoembraced by the invention. Specifically, because a plurality ofnucleotide sequences (or codons) encoding a single amino acid exist,there are a large number of nucleic acids encoding the amino acidsequence set forth in SEQ ID NO:1 in the Sequence Listing. These nucleicacids are thus embraced by the nucleic acids of this invention. As usedherein, “encode a protein” means that when DNA is double-stranded, theterm includes a DNA wherein either of the complementary two strands isprovided with the nucleotide sequence encoding the protein. Therefore,embraced by the invention are nucleic acids comprising the nucleotidesequences directly encoding the amino acid sequence set forth in SEQ IDNO:1 in the Sequence Listing and nucleic acids comprising the nucleotidesequences complementary to the foregoing nucleotide sequences.

Next, the farnesyl pyrophosphate synthase genes according to theinvention will be described.

The nucleic acid of this invention is one (cDNA) having the nucleotidesequence set forth in SEQ ID NO:3 in the Sequence Listing and encoding ahop farnesyl pyrophosphate synthase.

The nucleic acid of the invention may also be a part of the nucleotidesequence set forth in SEQ ID NO:3 in the Sequence Listing.

Further, the nucleic acid of the invention may be one that hybridizes tothe nucleotide sequence with 1029 bases set forth in SEQ ID NO:3 in theSequence Listing under stringent conditions and encoding a proteinpossessing the farnesyl pyrophosphate synthase activity; and itsnucleotide sequence is not particularly limited insofar as it satisfiesthese conditions. Still further, the nucleic acids of the inventionembrace nucleic acids having nucleotide sequences complementary to thatof the aforementioned nucleic acid undergoing hybridization understringent conditions. Specifically, there are mentioned, among others,nucleic acids having deletions of, substitutions of, insertions to oradditions to several bases of the nucleic acid having the nucleotidesequence of SEQ ID NO:3 and possessing the farnesyl pyrophosphatesynthase activity. As used herein, “deletion, substitution, insertion,or addition” includes not only a short deletion, substitution, insertionor addition with 1 to 10 bases, but also a long deletion, substitution,insertion or addition with 10 to 100 bases. The “farnesyl pyrophosphatesynthase activity” refers to the activity allowing the reaction toproceed by which farnesyl pyrophosphate is synthesized by the catalyticaction of the farnesyl pyrophosphate synthase. In this case thesubstance that serves as a substrate for the farnesyl pyrophosphatesynthase is not particularly limited insofar as it is a substance fromwhich farnesyl pyrophosphate can be ultimately synthesized.Specifically, there are mentioned isopentenyl pyrophosphate and geranylpyrophosphate.

The geranyl pyrophosphate and farnesyl pyrophosphate are regarded in ahop as the precursors of essential oil constituents such as myrcenes,humulenes, caryophyllenes, and farnesenes. Thus, by detecting a nucleicacid having the nucleotide sequence set forth in SEQ ID NO:3 in theSequence Listing, it will be possible to utilize the nucleic acid as agenetic marker for the traits concerning the regulation of the metabolicsystem for hop's essential oil constituents and the essential oilconstituents themselves. The detection of a nucleic acid does notrequire the whole nucleotide sequence set forth in SEQ ID NO:3 in theSequence Listing; for example, part of the sequence may be amplified byPCR and the detection may be carried out by gene analysis techniquessuch as nucleotide sequencing (base sequencing) or RFLP (RestrictionFragment Length Polymorphism). Therefore, the nucleic acids of thisinvention embrace nucleic acids comprising a part of the nucleotidesequence set forth in SEQ ID NO:3 in the Sequence Listing.

Next, the promoter region of the farnesyl pyrophosphate synthase geneaccording to this invention will be described.

The nucleic acid of this invention is a nucleic acid having thenucleotide sequence set forth in SEQ ID NO:2 in the Sequence Listing.The nucleic acid of the invention may be part of the nucleic acid havingthe nucleotide sequence with 4699 bases, as set forth in SEQ ID NO:2 inthe Sequence Listing, or alternatively it may be the nucleotide sequencerepresented by base no. 1 to no. 1886.

The nucleic acid having the nucleotide sequence set forth in SEQ ID NO:2is genomic DNA of farnesyl pyrophosphate synthase and the nucleotidesequence encoding the starting methionine of farnesyl pyrophosphatesynthase is from base no. 1887 to no. 1889 within the genomic DNA. Thus,base no. 1 to no. 1886 in the nucleotide sequence set forth in SEQ IDNO:2 in the Sequence Listing is a 5′-noncoding region, within which thepromoter region of the farnesyl pyrophosphate synthase gene iscontained. It will be difficult to unambiguously define the boundariesof both ends of the promoter region within base no. 1 to no. 1886;therefore, the nucleic acids of this invention embrace sequencescomprising part of base no. 1 to no. 1886 insofar as they possess thepromoter activity.

The nucleic acid of this invention may be one that hybridizes to thenucleic acid having the nucleotide sequence set forth in SEQ ID NO:2 orto the nucleic acid having the nucleotide sequence represented by baseno. 1 to no. 1886 within the foregoing nucleic acid under stringentconditions and that possess the promoter activity; and its nucleotidesequence is not particularly limited. The nucleic acids of thisinvention further embrace nucleic acids having the nucleotide sequencecomplementary to that of the nucleic acid that undergoes hybridizationunder stringent conditions and that possesses the promoter activity.Specifically, there are mentioned, among others, nucleic acids havingone or more deletions in, substitutions in, insertions to or addition tothe nucleic acid having the nucleotide sequence of SEQ ID NO:2 andpossessing the promoter activity. As used herein, “deletion,substitution, insertion, or addition” includes not only a shortdeletion, substitution, insertion or addition with 1 to 10 bases, butalso a long deletion, substitution, insertion or addition with 10 to 100bases.

Next, the preferred method of isolating the nucleic acids of thisinvention and analyzing the functions of their gene products will bedescribed.

The nucleic acid of the invention can be isolated through steps (1) to(5) as described below and it is possible to confirm in steps (6) to (7)that the isolated gene will display the farnesyl pyrophosphate synthaseactivity or will possess the promoter activity.

1. Isolation of Farnesyl Pyrophosphate Synthase Gene and its Promoterfrom a Hop

(1) Preparation of Hop Genomic DNA

The preparation of the hop genomic DNA can be carried out according to amethod known in the art: for example, the method of Wagner, D. B. et al.(Proc. Natl. Acad. Sci. USA 84, 2097-2100 (1987)) can be followed.

(2) Isolation of Farnesyl Pyrophosphate Synthase Gene and its promoter

Partial fragments of the farnesyl pyrophosphate synthase gene can beisolated by designing primers based on the known nucleotide sequences ofthe farnesyl pyrophosphate synthase genes from other plants such asArabidopsis and corn and by using the known methods such as Inverse PCRor Cassette-ligation mediated PCR. As Inverse PCR is shown in theschematic diagram of FIG. 2, DNA that serves as a sample is digestedwith restriction enzymes; the restriction enzyme-digested product isthen cyclized to a sample that serves as a template prior toamplification; and primers synthesized in directions opposite to thosefor the primers to be used in ordinary PCR are used to perform PCR. Themethod makes it possible to amplify the upstream or downstream regionadjacent to a specific nucleotide sequence. As a concrete example ofInverse PCR, there may be mentioned the method of Liu, Y. G. et al.(Genomics 25, 674-681 (1995)). As schematically shown in FIG. 3,Cassette-ligation Mediated PCR is a method to be used when an unknownnucleotide sequence adjacent to a known nucleotide sequence will bedesirably determined (e.g., the method as described in the protocolattached to a Takara LA PCR in vitro Cloning Kit (Takara Shuzo Co.Ltd.)). Specifically, the nucleic acid containing such nucleic acidregion is first digested with restriction enzyme. Adapter nucleic acidsof known nucleotide sequences having the restriction enzyme recognitionsites from which primers can be designed are ligated to theaforementioned nucleic acid. Then, the unknown nucleotide sequenceregion flanked by the known sequence regions is amplified by PCR and theamplified product may be sequenced. By repeating such Inverse PCR and/orCassette-ligation Mediated PCR, it is possible to isolate the entireregion of the hop farnesyl pyrophosphate synthase gene and its promoter.

(3) Sequencing

The isolated genes can be sequenced by a method known in the art: forexample, it can be done by following the protocol attached to an ABIPRISM Dye Primer Cycle Sequencing Ready Reaction Kit available from PEBiosystems Inc. The nucleotide sequences determined by theaforementioned method can be subjected to homology search using adatabase such as http://www.ncbi.nlm.nih.gov/BLAST and thus it will bepossible to find out the presence or absence of homology with the knowngenes obtained from other species of plants as well as the degree ofhomology. This will enable determination as to whether or not theobtained genes are novel.

(4) Preparation of Total RNA from the Respective Tissue Fractions

After an arbitrary fraction has been prepared, the preparation of totalRNA can be carried out according to a method known in the art: forexample, the method of Chang, S. et al. (Plant Molecular Biology Report11, 113-116 (1993)) can be followed.

(5) Isolation of Farnesyl Pyrophosphate Synthase Gene cDNA

cDNA of the farnesyl pyrophosphate synthase gene can be isolatedaccording to a method known in the art: specifically, primers may bedesigned based on the nucleotide sequence of the genomic DNA for thefarnesyl pyrophosphate synthase gene isolated in (2) and cDNAsynthesized from the total mRNA may be used as a template to effectisolation through RT-PCR. For a concrete method in this case, the methodas described in the protocol attached to a Titan One Tube RT-PCR Systemavailable from Roche Diagnostics Inc. can be employed, for example.

(6) Functional Analysis of the Protein Encoded by the Isolated FarnesylPyrophosphate Synthase Gene

The protein encoded by the farnesyl pyrophosphate synthase gene isolatedin (5) can be expressed in E. coli cells by incorporating cDNA of thefarnesyl pyrophosphate synthase gene into an expression vector andintroducing the vector into an E. coli cell. The expression andpurification of the protein encoded by the farnesyl pyrophosphatesynthase gene described above can, for example, be carried out by themethod as described in the protocol attached to a QIAexpress ExpressionSystem (QIAGEN Inc.). The functions of the farnesyl pyrophosphatesynthase protein expressed in those E. coli cells and purified can beconfirmed by a method known in the art: for example, the method ofSylvie A. et al. (Arch. Biochem. Biophys. 321, 493-500, (1995)) can beused for confirmation.

(7) Northern Hybridization (Hereunder Referred to as “NorthernAnalysis”)

The isolated farnesyl pyrophosphate synthase gene can be used as a probeand Northern analysis can be done to analyze as to in which tissue theisolated farnesyl pyrophosphate synthase gene is expressed, or as to inwhich tissue the isolated farnesyl pyrophosphate synthase functions. Forexample, the analysis can be done by the method as described in “The DIGSystem User's Guide for Filter Hybridization” (Boeringer Manheim) p.53-55 (1995).

Next, one embodiment that is made possible by the nucleic acid of thisinvention will be described.

(1) Probes for Use in Hybridization

By using a part or the whole of the nucleotide sequence disclosed inthis invention as a hybridization probe, it is possible to at leastdetect the farnesyl pyrophosphate synthase gene expressed in a hop. Byusing a part or the whole of the nucleotide sequence disclosed in thisinvention as a hybridization probe to investigate the gene expression inhop tissues, it is also possible to identify the distribution of thegene expression.

When a part or the whole of the nucleotide sequence disclosed in thisinvention is used as a hybridization probe, the method of hybridizationitself is not particularly limited; however, there are specificallymentioned as examples, Northern hybridization, Southern hybridization,colony hybridization, dot hybridization, Fluorescence in situhybridization (FISH), in situ hybridization (ISH), DNA chip method, andmicroarray method.

When the nucleotide sequence of this invention is used as thehybridization probe, the nucleotide length (base length) of at least 20bases is necessary; and a gene having a nucleotide length of 20 or moreconsecutive bases within the gene sequence of the invention ispreferably used. There are used more preferably, one having a nucleotidelength of 40 or more bases, and most preferably one having a nucleotidelength of 60 or more bases.

The nucleic acid probe technique is well known to one skilled in theart, and suitable hybridization conditions for the probes withindividual lengths according to this invention and the objectivepolynucleotide can readily be determined. To obtain hybridizationconditions optimal for the probes containing varying lengths, suchmanipulations are well known to one skilled in the art. For example,Sambrooks et al., “Molecular Cloning: A Laboratory Manual,” 2nd. Ed.,Cold Spring Harbor (1989) may be referred to.

Here, the probes are preferably labeled so that they can be easilydetected. Detectable labels may be any type or a portion thereof thatcan be detected either by the naked eyes or with a device. Thedetectable labels that are ordinarily used are, for example, radioactivelabels such as ³²P, ¹⁴C, ¹²⁵I, ³H, and ³⁵S. Biotin-labeled nucleotidescan be incorporated into nucleic acids by using nick translation,chemical and enzymatic means, or the like. The biotin-labeled probes aredetected after hybridization utilizing labeling means such asavidin/streptavidin, fluorescent labels, enzymes, and gold colloidalcomplexes. The nucleic acids may be labeled by being bound to proteins.Alternatively, there may be used nucleic acids that have cross-linked tothe radioactive or fluorescent histone single strand biding protein.

(2) Primers for Use in PCR

It is also possible to detect the farnesyl pyrophosphate synthase geneby using as primer any sequence of the disclosed nucleotide sequence andthe Polymerase Chain Reaction (PCR) method. For example, RNA can beextracted from a sample to be assayed and the gene expression can besemi-quantitatively determined by RT-PCR. Such a method can be carriedout by a technique well known in the art.

When the nucleic acid of this invention is used as a PCR primer, thenucleotide length of from 10 to 60 bases is necessary; and a nucleicacid having a nucleotide length of from 10 to 60 consecutive bases (morepreferably 15 to 30 bases) within the nucleic acid of the invention ispreferably used. Generally, the GC content of the primer sequence ispreferably from 40 to 60%. Further, it is preferred that there be nodifference in Tm value between two primers. It is also preferred thatannealing do not take place at the 3′-ends of the primers and asecondary structure do not occupy within the primers.

(3) Screening for Nucleic Acids

It is possible to detect the distribution of expression of the farnesylpyrophosphate synthase gene that is expressed in a hop by using a partor the whole of the nucleotide sequence disclosed in this invention. Forexample, the part or the whole of the nucleotide sequence disclosed inthe invention can be used as a hybridization probe or as a primer in PCRto detect the distribution of gene expression.

DNA chips, microarrays or the like can also be used to detect saiddistribution of gene expression. Specifically, parts or the whole of thenucleotide sequence disclosed in this invention can all be applied ontothe chip or the array directly. RNA extracted from cells is labeled witha fluorescent substance or the like and is hybridized to the chip or thearray; it is then possible to analyze as to in which cell the gene ishighly expressed. DNA applied onto the chip or the array may be a PCRproduct obtained by using the part or the whole of the nucleotidesequence disclosed in this invention.

(4) DNA Cloning

It is possible to clone a gene that is expressed at least in a hop byusing a part or the whole of the nucleotide sequence disclosed in thisinvention. For example, the part or the whole of the nucleotide sequencedisclosed in the invention can be used as a probe in Northernhybridization or in colony hybridization, or as a primer in PCR to clonethe part or the whole of the nucleotide sequence disclosed in thisinvention.

In embodiments other than those described above, it will be possible toobtain information on the hop farnesyl pyrophosphate synthase or tocarry out the transformation of a hop and the production of itssecondary metabolic products.

Specifically, the farnesyl pyrophosphate synthase mentioned above is anenzyme involved in the metabolism of isoprenoids on which a variety ofsubstances in plants (such as pigments, odorants, phytohormones,phytoalexins and defense substances against pests) are based. Therefore,by using the farnesyl pyrophosphate synthase genes isolated as mentionedabove, it will be possible to control the plant metabolic systems forpigments, odorants, phytohormones, phytoalexins, and defense substancesagainst pests and to detect the genes responsible for these traits.

It will also be possible to produce the secondary metabolites of plantsin vitro by using the farnesyl pyrophosphate synthase produced throughgene manipulations using the farnesyl pyrophosphate synthase genesisolated in this invention.

There appears to be the possibility that farnesyl pyrophosphate synthaseis involved in the metabolic systems of a hop for hop resin (hop resinconstituents) and xanthohumol (Brauwelt, 36, 1998) the latter of whichis said to possess anticancer action. It will further be possible tocontrol the metabolic systems for the hop resin and xanthohumol by usingthe nucleic acids of this invention as well as to utilize said nucleicacids as genetic makers for the hop resin and xanthohumol.

Accordingly, it will be possible to carry out the method of breeding ahop through gene manipulations that conventionally have had to rely onexperience and intuition. For example, a plant transformation techniqueis utilized to introduce the farnesyl pyrophosphate synthase gene ofthis invention into a hop. It will thus be possible to control thecomposition of the secondary metabolites in luplin gland. Accordingly,it will become possible to improve and maintain the qualities of foodsutilizing hops (e.g., beer and low malt beer) as well as to improve andmaintain the qualities of drugs utilizing the secondary metabolites.

BY utilizing the nucleic acid containing the promoter region of thefarnesyl pyrophosphate synthase gene according to this invention, a geneto be desirably introduced into an objective hop and a terminatorcapable of functioning in the hop are linked to the downstream of thepromoter, and this is introduced to the hop. The aforementioned genewill thus be allowed to be specifically expressed in the luplin gland.

EXAMPLES

This invention will be described more concretely by referring to theexamples; however, the invention is not to be limited by these examples.

Example 1

Preparation of Hop Genomic DNA

The preparation of hop genomic DNA was carried out in the followingmanner described. Specifically, the leaves of a hop was freeze-ground inliquid nitrogen, suspended in a 2% CTAB solution [2%cetyltrimethylammonium bromide, 0.1 M Tris (pH 9.5), 20 mM EDTA, 1.4 MNaCl, 5% β-mercaptoethanol], and incubated at 65° C. for 30 minutes.After the suspension was extracted with chloroform/isoamyl alcohol(24:1) twice, DNA and RNA was allowed to precipitate by adding a ¾-foldamount of isopropanol. The precipitated DNA and RNA was dissolved in aHigh Salt TE buffer [1M sodium chloride, 10 mM Tris (pH 8.0), 1 mM EDTA]and it was incubated with addition of RNase at 60° C. to decompose onlyRNA. To this was added a two-fold amount of isopropanol, resulting inthe precipitation of DNA. The precipitated DNA was washed with 70%ethanol and was then dissolved to prepare a genomic DNA sample.

Example 2

Isolation of Farnesyl Pyrophosphate Synthase Gene and its Promoter

Out of the amino acid sequences of farnesyl pyrophosphate synthase forArabidopsis, corn, guayule, Hevea, white lupin, and pepper whosenucleotide sequences were known, the sequence that was consensus amongthe respective plants was made a basis on which Primer 1 (SEQ ID NO:4)and Primer 2 (SEQ ID NO:5) were synthesized. These primers were usedtogether with the hop genomic DNA as a template to carry out PCR,producing Fragment 1 in FIG. 1.

Next, the resulting amplified fragment was sequenced. Primer 3 (SEQ IDNO:6), Primer 4 (SEQ ID NO:7), Primer 5 (SEQ ID NO:8), and Primer 6 (SEQID NO:9) shown in Table 1 were designed based on the obtained nucleotidesequence, and Inverse PCR was performed to obtain Fragments 2 and 3 inFIG. 1.

TABLE 1 SEQ ID Primer No. Nucleotide sequence 1 45′-GGYTGGTGYATTGAATGG-3′ 2 5 5′-TAAAAYGARTARTARGCHGTYTT-3′ 3 65′-CCTTTGGTACTCTAAACCAGCAGGG-3′ 4 7 5′-TTACAAAGTGTTAAAAGGGTATCCC-3′ 5 85′-AGGTGGAATTCCAAACAGCCTCGGG-3′ 6 9 5′-TTTGATCACCACAATTGAAGGAGAG-3′ 7 105′-GACATTGTAATCCAGCATCTGC-3′ 8 11 5′-CACAGAGAAATTGAACTTGGTC-3′ 9 125′-CACTTCCTTTGACCTGTTTG-3′ 10 13 5′-AAGCTCGTGGAGTAACCCTC-3′ 11 145′-GCGTGTTTGCGGATTACGAG-3′ 12 15 5′-TGAGAAGGATTTTGGCAGCC-3′ 13 165′-GAATTCTTATGATTAACCAAAAAC-3′ 14 175′-CGGGATCCATGAGTGGTTTAAGGTTCAAAAT-3′ 15 185′-CGGGATCCTTACTTCTGCCTCTTGTAGATC-3′

Specifically, the hop genomic DNA was digested with restriction enzymesBglII or HindIII. A DNA Ligation Kit Ver. 1 (Takara Shuzo Co. Ltd.) wasused to carry out self-ligation following the protocol attached thereto.After the self-ligation was completed, a portion of the reactionsolution was used as a template to carry out PCR by employing Primers 3and 5. Subsequently, a portion of the reaction solution for which thePCR had been completed was used as a template to carry out PCR byemploying Primers 4 and 6 shown in Table 1. Fragments 2 and 3 in FIG. 1were thus obtained.

Similarly, Primer 7 (SEQ ID NO:10), Primer 8 (SEQ ID NO:11), and Primer9 (SEQ ID NO:12) were designed based on the nucleotide sequence ofFragment 2 in FIG. 1. The hop genomic DNA digested with restrictionenzyme EcoRI was subjected to self-ligation, and this was used as atemplate to carry out PCR again by employing Primers 7 and 9 describedabove. Fragment 4 in FIG. 1 was thus obtained and it was sequenced.

Fragment 5 in FIG. 1 was isolated with a Takara LA PCR in vitro CloningKit (Takara Shuzo Co. Ltd.) by Casette-ligation mediated PCR accordingto a protocol attached thereto. Specifically, the hop genomic DNA wasdigested with restriction enzyme EcoRI and to this was linked an EcoRIadapter included in the kit. PCR was next carried out by using Primer 10(SEQ ID NO:13) that had been designed on the basis of the nucleotidesequence of Fragment 3 and cassette primer C1 included in the kit. ThisPCR reaction solution was further used as a template to carry out PCR byemploying Primer 11 (SEQ ID NO:14) that had been designed on the basisof the nucleotide sequence of Fragment 3 and cassette primer C2 includedin the kit. Fragment 5 was thus obtained and it was sequenced.

Finally, PCR was carried out by using the hop genomic DNA as a templateand Primer 12 (SEQ ID NO: 15) and Primer 13 (SEQ ID NO: 16) that hadbeen designed on the basis of Fragment 4 and Fragment 5 in FIG. 1,respectively. Fragment 6 was thus obtained that contained the hopfarnesyl pyrophosphate synthase gene and a promoter thereof. All the PCRmanipulations described above were carried out using an ExpandHigh-Fidelity PCR System (Boeringer Manheim AG) according to theprotocol attached thereto.

Example 3

Sequencing of the Hop Farnesyl Pyrophosphate Synthase Gene and itsPromoter

Both ends of Fragment 6 containing the hop farnesyl pyrophosphatesynthase gene and its promoter that had been obtained in Example 2 weremade blunt by using a Takara BKL Kit (Takara Shuzo Co. Ltd.) and it wassubcloned into a pUC vector. The protocol attached to the kit wasfollowed to make both ends of Fragment 6 blunt and to effect thesubcloning into the pUC vector.

Sequencing was carried out using an ABI PRISM Dye Terminator CycleSequencing Ready Reaction Kit (ABI373S type available from PE BiosystemInc.) according to the protocol attached thereto. The nucleotidesequence of Fragment 6 is shown in SEQ ID NO:2 in the Sequence Listing.

Example 4

Preparation of the Respective Tissue Fractions and Total RNA

The leaves, stem, luplin (−), and luplin (+) fractions of a hop wereprepared for the tissues from which total RNA would be extracted. Asused herein, “luplin (−) fraction” was a fraction principally recoveredfrom the bract of a cone where luplin gland was hardly present. The“luplin (+) fraction” was a fraction consisting principally of luplingland that was obtained by removing from the cone, tissues other thanthe luplin gland as much as possible. These tissue fractions werefreeze-ground in liquid nitrogen, suspended in a 2% CTAB solution [2%cetyltrimethylammonium bromide, 0.1 M Tris (pH 9.5), 20 mM EDTA, 1.4 MNaCl, 5% β-mercaptoethanol], and incubated at 65° C. for 10 minutes.After the suspension was extracted with chloroform/isoamyl alcohol(24:1) twice, a ⅓-fold amount of 10M lithium chloride was added to theextract and it was allowed to stand overnight. After centrifugation at15,000 rpm for 10 minutes, the precipitates are dissolved in water. Whentotal RNA was to be used in Example 5, the precipitates were dissolvedin DNase reaction buffer [100 mM sodium acetate (pH 5.2), 5 mM magnesiumchloride] in place of water and it was incubated with addition of DNaseat 37° C. to decompose only DNA. To the solution was further added a⅓-fold amount of 10 M lithium chloride. It was allowed to standovernight and centrifuged at 15,000 rpm for 10 minutes. After washing,the precipitates were washed with 70% ethanol and were then dissolved inwater again to prepare a total RNA sample.

Example 5

Isolation and Sequencing of cDNA for the Farnesyl Pyrophosphate SynthaseGene

Both ends of the coding region for the hop farnesyl pyrophosphatesynthase gene were presumed that was set forth in SEQ ID NO:2 andsequenced in Example 3 based on the information about the farnesylpyrophosphate synthase gene of Arabidopsis (among others) or the likethe nucleotide sequence of which was known. Primers were designed thathad the sequences obtained by appending the BamHI recognition sequenceto those sequences and they were used with the total RNA produced inExample 4 as a template, where cDNA of the farnesyl pyrophosphatesynthase gene was isolated by RT-PCR. Specifically, Primer 14 (SEQ IDNO:17) and Primer 15 (SEQ ID NO:18) were used as primers and PCR wascarried out using a Titan One Tube RT-PCR System (Roche DiagnosticsInc.) according to the protocol attached thereto. The thus obtained cDNAof the farnesyl pyrophosphate synthase gene was subcloned into a PCR2.1vector (Invitrogen Inc.) to prepare pFPPS101R. The subcloned cDNA of thefarnesyl pyrophosphate synthase gene was sequenced using an ABI PRISMDye terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems Inc.)and a DNA sequencer ABI 373S type (PE Biosystems Inc.) according to theprotocol attached thereto. The nucleotide sequence of cDNA for theobtained farnesyl pyrophosphate synthase gene is shown in SEQ ID NO:3 inthe Sequence Listing. The amino acid sequence of the protein encoded bythis cDNA is shown in SEQ ID NO:1.

Within the nucleotide sequence set forth in SEQ ID NO:3, the 660th basediffered from the corresponding base (the 3737th base in SEQ ID NO:2) inthe genomic DNA, although confirmation of the nucleotide sequences wasconducted plural times. This is thought to be a base incorporation errorresulting from RT-PCR conducted when cDNA was isolated. However, thiserror does not affect the functional analysis of protein as will bedescribed in Example 6 because it leads to the amino acid identical tothat which should have been encoded at the amino acid level.

Example 6

Functional Analysis of the Protein Encoded by the Isolated FarnesylPyrophosphate Synthase Gene

In order to determine whether or not the protein encoded by the isolatedfarnesyl pyrophosphate synthase gene possessed the farnesylpyrophosphate synthase activity, cDNA of the farnesyl pyrophosphatesynthase gene isolated in Example 5 was treated with restriction enzymeBamHI to give DNA. The DNA was incorporated into the BamHI site of anexpression vector pQE30 [attached to a QIAexpress Expression System(QIAGEN Inc.)]. It was then introduced into E. coli to have the farnesylpyrophosphate synthase gene expressed in the E. coli cells and theexpressed product was purified. Expression of the farnesyl pyrophosphatesynthase gene in the E. coli cells and purification of the expressedproduct was carried out according to the protocol attached to aQIAexpress Expression System (QIAGEN Inc.).

Next, the method of Sylvie A. et al. (Arch. Biochem. Biophys. 321,493-500 (1995)) was followed to determine whether or not the obtainedexpression product possessed the farnesyl pyrophosphate synthaseactivity. Specifically, to 100 μl of enzyme reaction solution (50 mMTris-HCl, 2 mM dithioerythritol, 1 mM magnesium chloride, 100 μMdimethylallyl pyrophosphate) were added 2 μl (28 μg) of the purifiedexpression product of the farnesyl pyrophosphate synthase gene and 2.5μl (0.05 μCi) of ¹⁴C-isopentenyl pyrophosphate, and the reaction wasallowed to take place at 30° C. for 30 minutes. To alkaline phosphatase(Wako Pure Chemical Industries, Ltd.) was added 30 μl of 10-foldconcentration reaction buffer (as attached). Alkaline phosphatase, 1 μl,(10 units) was then added to the reaction, which was allowed to continueat 37° C. for 3 hours. Then, the reaction was further continued at 25°C. overnight. To the reaction solution was added 1 μl of farnesol (4.5nmol) as a carrier and further 200 μl of hexane, and upon mixing thehexane layer was recovered after centrifugation at 10,000 rpm for oneminute. Hexane, 100 μl, was again added to the remaining water layer andit was mixed and centrifuged to recover a hexane layer, which wascombined with the hexane layer recovered earlier. The hexane extract wasconcentrated to 1 μl by being blown with nitrogen gas. After 10 μl ofmethanol was added and mixed to the concentrate, 1 μl was spotted ontothin layer chromatography (HPTLC-aluminum sheets silica gel 60 F254pre-coated available from Merk KGaA) and developed in a developingsolvent (benzene:ethyl acetate=9:1). Farnesol and geraniol weresimultaneously spotted as a standard. After the thin layer chromatogramplate for which development had been completed was dried, iodine wassprayed onto the plate to ascertain the positions of farnesol andgeraniol. Exposure to a X-ray film was carried out at −80° C. for 7days. The obtained results are shown in FIG. 4.

As FIG. 4 shows, the signals of the reaction products were detected atthe positions of farnesol and geraniol. Specifically, it was confirmedthat the protein encoded by the isolated farnesyl pyrophosphate synthasegene possessed the farnesyl pyrophosphate synthase activity.

Example 7

Northern Hybridization

Northern hybridization was carried out to determine in which tissue of ahop the isolated farnesyl pyrophosphate synthase gene was expressed aswell as to determine the level of its expression.

Plasmid pFPPS101R prepared in Example 5 was first digested withrestriction enzyme KpnI to produce a linearized form. A DIG RNA labelingkit (SP6/T7) (Roche Diagnostics Inc.) was used to prepare a RNA probefor the farnesyl pyrophosphate synthase gene by employing the linearizedform as a template. The preparation method was performed according tothe protocol attached to the kit.

The total RNAs for the leaves, the stem, the luplin (−) fraction, andthe luplin (+) fraction that had been prepared in Example 4 (each 15 μl)were subjected to electrophoresis using denatured agarose gel (1.2%agarose, 6.7% formaldehyde, 20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA,pH 7.0). The gel for which electrophoresis had been completed was shakenin distilled water three times each for 40 minutes and afterformaldehyde in the agarose gel was removed, RNA in the agarose gel wastransferred to a nylon membrane by using 20×SSC (0.3 M sodium citrate, 3M sodium chloride, pH 7.0) as buffer. The nylon membrane to which RNAhad been transferred and the aforementioned probe were used to carry outhybridization at 68° C. overnight. Here, the composition of thehybridization buffer used in the hybridization was 5×SSC, 0.02% SDS,0.1% N-lauroylsarcosin, 50% formamide, and 2% Blocking Reagent (RocheDiagnostics Inc.). After hybridization, a detergent (0.1% SDS, 2×SSC)was used to carry out washing-treatment twice each at 68° C. for 30minutes; further, a detergent (0.1% SDS, 0.1×SSC) was used to carry outwashing-treatment twice each at 68° C. for 30 minutes. After washing,the RNA fragment to which the probe had been hybridized was detected.The detection was carried out according to the protocol described in“The DIG System User's Guide for Filter Hybridization” (Roche DiagnosticInc.). The results obtained are shown in FIG. 5.

From the results of FIG. 5, there was observed in each of the tissuefractions, the leave, the stem, the luplin (−) fraction, and the luplin(+) fractions a signal at the position of 1.1 kb approximating to thesize of the nucleic acid having the nucleotide sequence set forth in SEQID NO:2 in the Sequence Listing that encodes the farnesyl pyrophosphatesynthase gene. This confirmed that the hop farnesyl pyrophosphatesynthase gene was expressed in each of the tissue fractions. However,the intensity of the signal was in the order of the luplin (+)fraction>the luplin (−) fraction>the stem>the leaf. This confirmed thatthe degree of mRNA derived from the farnesyl pyrophosphate synthase geneoccupying in each tissue was the greatest in the luplin gland, nextplace in the stem and the bract, and the least in the leaf. In otherwords, it was confirmed that the farnesyl pyrophosphate synthase genewas expressed in the strongest manner in the luplin gland and thus thepromoter of the farnesyl pyrophosphate synthase gene had the strongestpromoter activity in the luplin gland.

INDUSTRIAL APPLICABILITY

As described above, it is possible to identify the farnesylpyrophosphate synthase proteins and genes according to this invention.It will, therefore, reveal the genes involved in the biosynthesis ofsecondary metabolites in a hop as well as the nucleotide sequences ofthe promoter genes that function in the luplin gland of the hop in atissue-specific manner. This will allow for the transformation of thehop by gene manipulations and the in vitro synthesis of the hopsecondary metabolites.

1. An isolated nucleic acid encoding a protein comprising the amino acidsequence of SEQ ID NO:
 1. 2. An isolated nucleic acid comprising thenucleotide sequence of SEQ ID NO:
 3. 3. An isolated nucleic acidcomprising the nucleotide sequence of SEQ ID NO:
 2. 4. An isolatednucleic acid comprising nucleotides 1-1886 of SEQ ID NO:
 2. 5. Anisolated nucleic acid which hybridizes to nucleotides 1 to 1886 of SEQID NO: 2 or to a fully complementary nucleic acid thereof understringent conditions, wherein said nucleic acid possesses promoteractivity, and wherein the stringent conditions comprise hybridization in6.0×SSC at 45° C. and washing in 0.1×SSC at 65° C.